Abstract
We believe that biopesticides should be used to solve the health
and ecological problems caused by chemical pesticides, but existing
biopesticides are not good enough. So, we decided to improve the
virulence of a fungal biopesticide. To evaluate the viability and to
improve our project, we communicated with stakeholders, including
small-scale farmers, agricultural companies, genetic engineering
experts, biosafety experts, and policymakers. We collected feedback
from multiple perspectives such as market demand, technical
solutions, safety considerations, legal viability, etc. All of this
allows us to optimize our designs and make them more realistic. As a
result, we decided to improve the virulence of a biopesticide and
include a suicide switch to ensure safety. The biosafety risks of
engineered biopesticides are our key concern, and we are still in
the process of prudent research. Click to see our mind map.
1. Introduction
Chemical pesticides are overused around the world in agriculture
and horticulture, which pose risks to the farmers, the customers,
and the non-target animals. Yearly, more than 26 million people
suffer from pesticide poisoning with nearly 220,000 deaths around
the world (Ansari, Moraiet, and Ahmad, 2013). In China, more than
two million tons of insecticides were disseminated in the
environment in 2020, killing native species and resulting in an
ecological disaster.
Pesticide use by each country in 2020 from Our World in Data.
Biopesticides are safer for human beings and other non-target
animals. They are relatively more eco-friendly. And they cause
little or no problem with post-harvest contamination (Fenibo et al.,
2021). However, certain limitations including low effectiveness and
limited availability make biopesticides unpopular compared to
chemical pesticides for farmers. To overcome the limitations,
genetic modification has become one promising method to improve the
effectiveness and efficiency of biopesticides.
Our project aimed to create a more eco-friendly, safer biopesticide
for farmers and growers to achieve sustainable development. Before
beginning, we first investigated the current use of biopesticides
and the potential commands of current users.
2. Biopesticide Market Research
To understand the current use and demand for biopesticides in the
market, we conducted interviews and surveys with pesticide users. We
found that pesticide users could be divided into two categories: the
small-scale farmers, and the agricultural companies, and interviewed
both groups. The feedback showed that small-scale farmers were more
concerned about cost and effectiveness and therefore mostly used
chemical pesticides. On the other hand, fruit growers and
agricultural companies preferred to use biopesticides. However, they
were concerned about the efficiency and safety of biopesticides.
According to the feedback, it was clear that there is a market
demand for biopesticides, and that fruit growers and agricultural
companies would be our main target users. It was also clear that the
goals of our project were to improve insecticidal efficiency and
ensure safety. During the interviews, we found Metarhizium, which became the fungus of our choice.
2.1 Small-scale farmers
To find out the current pesticide use of small-scale farmers, we
surveyed 69 farmers. The results showed that small-scale rice
growers preferred chemical pesticides due to concerns about cost and
insecticidal efficiency. Fruit growers, on the other hand, were less
price-sensitive and would be more willing to use biopesticides.
However, efficiency is still the core concern. During the
interviews, we also found that the widely used Metarhizium pesticide had a problem with efficiency.
Survey
Participants:
68 villagers (37 males and 31 females, ages ranging from 15 to 72
years old) from Yaojia Village, Haining, Zhejiang, China. Most of
them are smallholders whose household land is smaller than one acre.
Most of them are rice farmers.
Results and takeaways:
1) All 68 participants used pesticides in agriculture, and only 9%
used biopesticides.
2) One-third of the participants knew what biopesticides were.
3) Beauveria bassiana and Bacillus thuringiensis were the most famous biopesticides and one-fifth of
respondents also had heard of Materhizium before.
4) The participants believed that effect (81%) and cost (69%) were the
two most important factors of pesticides, which showed that our
choice to increase the virulence of M. anisopliae was correct.
5) 88% of the respondents agreed that biopesticides could be good
substitutes for chemical counterparts.
Interview
Interviewee:
Yanfang Wang
Yaojia Village, Haining, Zhejiang, China
Strawberry farmer who owned five plastic greenhouses
Suggestions and takeaways:
1) Compared to staple crops, fruits are more susceptible to pests.
2) Compared to the effectiveness of the pesticides, the cost is not of
the first concern because strawberries were profitable. Paying for a
better pesticide could increase the total profit for her.
3) Yanfang would try to use our product if it was very effective and
got approved.
2.2 Agricultural Companies
We then turned to the agricultural companies, which were bigger
customers and represented a broader market. Compared to
smallholders, agricultural companies were more cautious in
decision-making and we were eager to hear their voices. We
interviewed the managers of Shuangma and Shouguang Agricultural.
Both Shuangma and Shouguang Agricultural showed interest in our
engineered fungal biopesticide, which indicated a bright perspective
of our project. We concluded that large agricultural companies were
more likely to use our product compared with smallholders because
the companies were less sensitive to cost and had more concerns
about ecological safety, employee health, export, etc. Moreover,
concerns by Shuoyang about biosafety revealed the flaw of our
project. We realized that since our product's intended use was in
agriculture, our engineered fungus would inevitably be released into
the environment. We needed to develop a method to restrain the
reproduction and spread of the fungus without influencing its
killing ability against pests. After literature research, we decided
to incorporate a suicide switch into M. anisopliae. We found that the 2016 NYMU_Taipei iGEM Team designed a
light-induced suicide switch for M. anisopliae and we decided to improve it.
Interview
Interviewee:
Shuoyang Zhu
General Manager of Shuangma Agricultural Co., Ltd., Hefei, Anhui,
China. Shuangma holds over 100 acres of land.
Suggestions and takeaways:
1) They were already using M. anisopliae, and therefore would definitely try our improved version.
2) One disadvantage of fungal and bacterial biopesticides is that they
can reproduce in the environment and it is difficult to control.
3) We should evaluate the ecological safety of our engineered fungus,
especially its potential impacts on native species.
Interview
Interviewee:
Hongfeng Xu
Manager of Shandong Shouguang Agricultural Group. Shouguang has the
largest vegetable-producing base in China.
Suggestions and takeaways:
1) They had used M. anisopliae before but were not satisfied with its efficiency. They hoped
that our project could improve it.
2) He believed that the most important advantage of biopesticide was
its safety for humans. The health of the employees is the company's
top concern.
3) Biopesticides are especially suitable for exporting vegetables
because vegetable-importing countries may have strict regulations on
chemical pesticide residuals.
3. Safety Considerations
Through the surveys and interviews, we determined the goal of our
project, which was to develop a more efficient and safer
biopesticide. We identified a safe insect-specific toxin, which is
harmless to mammals, as the key to increasing the virulence of
fungal biopesticides against pests. When talking to some of the
interviewees and the iGEM Safety and Security Committee, we received
feedback on concerns about biosafety, especially about the toxin,
and the potential impacts on native insects and ecosystems. To ensure safety, we decided to include a suicide switch. We also planned future
biosafety tests including the survival and competitive ability
tests, horizontal gene transfer tests, etc.
3.1 LqhIT2 property and toxicity
When trying to find a toxin to increase the virulence of M. anisopliae, we realized that we needed to find a toxin that is only toxic to
insects but not mammals. We looked at the natural predators of
insects. The Israeli yellow scorpion (Leiurus quinquestriatus hebraeus) feeds on insects and produces potent venom against its prey. Some
ingredients of the venom are insect-specific, including LqhIT2.
LqhIT2 is a 61 amino acid-long depressant toxin. Researchers have
proven that LqhIT2 is only toxic to insects. LqhIT2 has been studied since the 1990s. Many experiments and
dozens of papers have confirmed that it is an insect-specific toxin.
One of the most convincing experiments was done by Zlotkin et al. in
1991. They injected purified LqhIT2 into live mice, and it showed no
toxicity. This experiment was repeated by Herrmann et al. in 1995,
and their results are consistent. The most recent research on LqhIT2
was published by Zhu et al in 2023. They analyzed the molecular
structure of LqhIT2 and compared it with other toxins. They
concluded that the channel residue and cavity shape of LqhIT2
determine its species selectivity of insects.
When submitting the check-in forms, the iGEM Safety and Security Committee raised concerns about LqhIT2. For our project to proceed safely, we sought instruction from The Center of Biosafety Research and Strategy at Tianjin University. This Center is the only research institution specializing in biosafety in China. Prof. Wang from the Center helped to assess the biosafety of our project, especially the toxin.
Assessment
Assessor:
Professor Fangzhong Wang
Center of Biosafety Research and Strategy, Tianjin University
Field of research: Biosafety evaluations
Suggestions and takeaways:
1) We should make sure LqhIT2 is not toxic for humans if it mutates.
2) We should test or at least plan to test the survival and competitive
ability of the engineered fungus.
3) We should test or at least plan to test the efficiency of the
suicide switch under different weather conditions.
4) We should test or at least plan to test the horizontal gene transfer
rate of the engineered fungus.
|
Gene |
Query Cover |
E value |
Per. Ident |
Accession |
Toxicity |
|
LqqIT2 |
98% |
4.00E-33 |
88.52% |
P19855.2 |
Insect-specific |
|
LqhIT5 |
98% |
9.00E-23 |
73.77% |
P81240.1 |
Insect-specific |
|
BotIT4 |
98% |
8.00E-34 |
95.08% |
P55903.1 |
Insect-specific |
|
BotIT5 |
98% |
3.00E-33 |
93.44% |
P55904.1 |
Insect-specific |
|
BotIT6 |
98% |
1.00E-18 |
63.93% |
P59864.1 |
Insect-specific |
|
BjIT2 |
98% |
1.00E-28 |
78.69% |
P24336.1 |
Insect-specific |
|
BmKITa |
98% |
4.00E-29 |
81.97% |
Q9XY87.1 |
Insect-specific |
|
BmKITb |
98% |
3.00E-28 |
80.33% |
Q95WX6.1 |
Insect-specific |
|
BmKITc |
98% |
2.00E-24 |
75.41% |
Q9Y1U3.2 |
Insect-specific |
|
BmKAEP |
98% |
2.00E-29 |
80.33% |
P15228.2 |
Insect-specific |
|
BmKAEP2 |
98% |
1.00E-28 |
81.97% |
Q86M31.1 |
Insect-specific |
|
BmKIT2 |
98% |
1.00E-28 |
81.97% |
P68727.1 |
Insect-specific |
|
BmKIT3 |
98% |
8.00E-29 |
83.61% |
Q17231.2 |
Insect-specific |
(Moskowitz et al., 1998; Li et al., 2000; Ali et al., 2001;
Goudetet al., 2002; Peng et al., 2002)
3.2 Impacts on native insects
As mentioned by Shuoyang and the Safety and Security Committee, our engineered biopesticide could kill native insects and thereby influence the ecosystem. After discussing with our instructors and advisors, we had to admit that ecological risk will always exist. However, we believe that our biopesticide will cause less harm to native insects and the ecosystem than chemical pesticides do.
Since our goal is to replace chemical pesticides with better
biopesticides, our biopesticides should at least work as well as
chemical pesticides. All the currently popular chemical pesticides
are broad-spectrum pesticides (organophosphates, pyrethroids,
neonicotinoids, etc.), which is understandable. If one pesticide can
only kill one species of pests (in that case it would not affect
non-target economically important insects), farmers would have to
prepare dozens of different types of pesticides to deal with
different pests. And farmers would also need to become entomologists
first to know which pesticide to use. So in the real world,
broad-spectrum pesticides are the only choices.
Although our fungal pesticide itself can infect native insects, it
should have a lesser impact on them compared to chemical pesticides.
First of all, unlike chemical pesticides, biopesticides tend to not
cause resistance (Fenibo et al., 2021). As a result, it should
require a lower pesticide dose. Secondly, some chemical pesticides
can remain in the environment for a year or more, accumulate in
animal bodies, and cause long-term harm to native insects (Ansari,
Moraiet, and Ahmad, 2013). Biopesticides, on the other hand, do not
accumulate. And finally, we plan to include a suicide switch into
our fungal pesticide. Our fungi will contain a photosensor that
leads to the inactivation of new-forming spores under the sunlight.
So our biopesticide will not have a long-term impact on native
insects.
Before commercialization, we will re-evaluate the impact of the
engineered biopesticide on the ecosystem by field trials.
3.3 Light-controlled suicide switch
To deal with the concerns of biosafety and to stop fungus spread in
the environment, we tried to find a proper mechanism for the fungus
to commit suicide after use. We found that the 2016 NYMU_Taipei iGEM Team designed a suicide
switch for M. anisopliae by ligating a KillerRed gene after a full-length Pmcl1
promoter. KillerRed is a red fluorescent protein that produces lethal
reactive oxygen species (ROS) upon exposure to light (Onukwufor et al., 2020). When the fungi invade the insect body, the hemolymph-inducible Pmcl1 promoter will start to express KillerRed. And when the fungi grow out of the insect body and try to spread
spores, their tissues are killed by sunlight. To improve the effectiveness of SuperNova and ROS, a nuclear
localization signal (NLS) derived from the SV40 T antigen should be
connected to the 3' end of the KillerRed sequence (Lu et al.,
2021). The SV40 NLS should guide the protein to be transported into
the cell nucleus, and let ROS attack the most vulnerable genomic DNA
(Paardekooper et al., 2019).
We planned to improve the suicide switch designed by the 2016
NYMU_Taipei iGEM Team. We found that compared to the full-length
Pmcl1 (2764bp), a truncated, shorter version of Pmcl1 (1586bp) can
lead to a twofold increase in downstream gene expression (Kanjo et
al., 2019). And Onukwufor et al. have proven that SuperNova can
produce three times as much ROS as KillerRed. As a result,
theoretically, a Pmcl1 (short) combined with SuperNova should be a
stronger suicide switch than the 2016 NYMU_Taipei iGEM Team's. To
verify our hypothesis and to find the strongest suicide switch, we
planned to test Pmcl1-SuperNova, Pmcl1(short)-SuperNova,
Pmcl1-KillerRed, and Pmcl1(short)-KillerRed.
3.4 Safety in labs
To minimize the risks of the experiments, we held a seminar with The Laboratory and Equipment Management Department of Zhejiang
University. To protect team members and the environment, we made
strict rules and adjustments to our experiment design.
As a filamentous fungus, M. anisolpliae produces spores. Spores are harmful to humans and are prone
to accidental leakage. We take measures to prevent spore spread. The
containers will only be opened in biosafety cabinets. When working
on our fungi, all the members will be required to wear nitrile
gloves and N95 masks. After each experiment, all the disposables
(gloves, pipette tips, etc.) will be autoclaved before disposal. UV
lamps in the biosafety cabinets will be turned on for at least 40
minutes after use.
To make sure no one is in direct contact with the toxin, we chose
not to extract or purify LqhIT2. And we will only insert the LqhIT2 gene downstream of an insect
hemolymph-specific promoter (Pmcl1). So LqhIT2 will only be
synthesized by our fungi in the insect bodies. Even if LqhIT2 leaked
from insect corpses, as a 61-amino acid peptide, it will be degraded
rapidly in the environment by microorganisms. Besides, it is
reported that plants are not capable of absorbing intact peptides
longer than five amino acids (Tegeder et al., 2010). As a result, it
is not possible that the toxin LqhIT2 will enter the crops and be
transferred to the human side.
3.5 Future biosafety tests
During the conversation, Prof. Wang mentioned the concerns about our engineered fungus impacting the environment. For our fungus to be proven safe, Prof. Wang listed the must-do tests of our project, including the survival and competitive ability tests, the suicide switch tests, and the horizontal gene transfer tests.
Although this year, we will only do experiments in the lab, and not do any field trials, we made plans for future tests. We planned two-stage tests. Stage one happens in artificial climate
chambers. We will introduce soil, Arabidopsis thaliana, and larvae of Galleria mellonella into the chambers. Stage two happens in experimental fields. We will measure each
factor and evaluate the biosafety.
4. Technical viability
As confirmed by literature research, we wanted to introduce
exogenous genes to the filamentous fungus. According to Peng & Xia,
2014, pBARGPE1 containing the bialaphos resistance gene (BarR) was
the appropriate vector. As for the transformation method, Peng and
Xia used microparticle bombardment. However, we were unable to
follow their path due to lack of equipment. Then, we found that Agrobacterium-mediated transformation worked for M. anisopliae (Duarte et al., 2007). So, our first instinct was to use Agrobacterium tumefaciens to transfer pBARGPE1 in our fungus. However, as high school
students, we were not experienced with molecular cloning, and we
were not sure about our design. So, we interviewed Prof. Zhu for
suggestions. Prof. Zhu pointed out that Agrobacterium-mediated transformation does not work with our plasmid, and we
should use protoplast transformation instead. To conclude, we could
use pBARGPE1 as the plasmid backbone and perform the protoplast
transformation to transform M. anisopliae. Our project's technical viability was confirmed and we were able
to proceed.
Interview
Interviewee:
Professor Xufen Zhu
Life Sciences Institute, Zhejiang University
Field of research: Microbiology and molecular biology
Suggestions and takeaways:
1) Our primary design was meant to fail because Agrobacterium-mediated transformation requires special shutter vectors.
2) We could switch to protoplast transformation instead without
changing our original vector choice.
3) The length of the inserted fragment should be taken into account
since a very long one would lower the efficiency of vector
construction, the rate of transformation, and the genetic
stability.
5. Legal viability
We plan to produce a genetically engineered fungal product, which
is strictly regulated in China. Therefore, we wanted to make sure
that our product at least has theoretical possibilities of entering
the market. And we wanted to know what regulations we needed to
comply with. After literature research, we discovered that the
genetically modified fungus has to undergo complicated tests before
entering the market, but it is possible. In 2023, 113 genetically
modified organisms received the Safety Certificate, and our M. anisopliae could be one in the future.
According to these regulations, we carefully tested the toxicity
and spreading capacity of the engineered M. anisopliae in our experiment. For detailed information, see Engineering Success.
Literature research
Results and takeaways:
1. According to the Measures for the Administration of the Safety
Evaluation of Agricultural Genetically Modified Organisms (2016
Revision, Article 13), our engineered fungus must go through three
stages of trials: intermediate test, environmental release test, and
production test.
2. During each stage, factors including genetic stability, potential
toxicity, spreading capacity, impacts on non-target organisms, etc.
will be evaluated (Appendix III. 1).
3. We should submit the Application for the Safety Evaluation of
Agricultural Genetically Modified Organisms to the State Commission
for the Safety of Agricultural Genetically Modified Organisms and
the Office for the Safety Management of Agricultural Genetically
Modified Organisms to start the evaluation process.
4. If approved, the Ministry of Agriculture and Rural Affairs of China
will issue the Agricultural Genetically Modified Organisms Safety
Certificate.
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